草鱼IFNa和IFNd重组蛋白表达、纯化及单克隆抗体制备  

Expression of recombinant proteins and preparation of monoclonal antibodies for IFNa and IFNd in grass carp(Ctenopharyngodon idella)

作　　者：王梓璇 贾钊 邬恺正 朱晓真 王俊亚 冯浩 邹钧 WANG Zixuan;JIA Zhao;WU Kaizheng;ZHU Xiaozhen;WANG Junya;FENG Hao;ZOU Jun(College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China;State Key Laboratory of Developmental Biology of Freshwater Fish,College of Life Sciences,Hunan Normal University,Changsha 410081,China)

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]湖南师范大学生命科学学院,省部共建淡水鱼类发育生物学国家重点实验室,湖南长沙410081

出　　处：《水产学报》2022年第11期2053-2065,共13页Journal of Fisheries of China

基　　金：国家自然科学基金(32030112)。

摘　　要：为系统研究草鱼I型干扰素的合成、分泌和免疫功能,本实验在大肠杆菌中表达并提纯了草鱼IFNa (Ci IFNa)和IFNd (Ci IFNd)重组蛋白。将Ci IFNa和Ci IFNd成熟肽分别克隆到pET-21d或pEHISTEVb表达载体上,并转化到大肠杆菌中;IPTG诱导表达获得Ci IFNa和Ci IFNd成熟肽的包涵体,经盐酸胍变性、蛋白复性和浓缩后,利用分子筛层析获得了纯度较高的重组蛋白。用重组蛋白免疫小鼠,通过PEG法诱导得到杂交瘤细胞;将稳定分泌抗体的阳性细胞株细胞悬液注射入小鼠腹腔,制备腹水抗体并进行纯化。本实验纯化了草鱼Ci IFNa和Ci IFNd各2株抗体,并采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)、酶联免疫吸附测定法(ELISA)、蛋白质印迹法(Western blot)和免疫荧光法对其进行了较全面的鉴定。实验表明,Ci IFNa和Ci IFNd单克隆抗体特异性好、效价高,能够特异识别在大肠杆菌和真核细胞中表达的重组蛋白,且不存在Ci IFNa和Ci IFNd分子间的交叉识别。本研究制备的单克隆抗体为深入研究草鱼干扰素的细胞来源和蛋白表达规律奠定基础。To understand the synthesis, secretion and immune functions of type I interferons(IFN) in Ctenopharyngodon idella, Ci IFNa and Ci IFNd were expressed in Escherichia coli(E. coli) cells and purified. The mature peptides of Ci IFNa and Ci IFNd were cloned into pET-21d and pEHISTEVb expression vectors, respectively, and transformed in E. coli Rosetta cells. The recombinant proteins were expressed as inclusion bodies after IPTG induction. Following denaturation with guanidine hydrochloride, renaturation and concentration, the recombinant proteins were purified by size exclusion chromatography and used for immunization of mice. Hybridoma cells were obtained using the PEG method and injected into the abdominal cavity of mice to generate ascites. Four monoclonal antibodies of Ci IFNa and Ci IFNd(2 antibodies each) were purified, and characterized by SDS-PAGE,ELISA, Western blot and immunofluorescence assay. It has been shown that the monoclonal antibodies produced had good specificity and high titers against the antigens and could specifically recognize recombinant proteins expressed in E. coli and eukaryotic cells. The Ci IFNa antibodies did not react with Ci IFNd and vice versa. Taken together, the Ci IFNa and Ci IFNd monoclonal antibodies prepared in this study may provide the basis for further indepth study of the cellular sources and protein expression profiles of IFNs in grass carp.

关 键 词：草鱼 干扰素 细胞因子 单克隆抗体 重组蛋白 

分 类 号：S942[农业科学—水产养殖]